Basic Residues at Position 11 of -Conotoxin LvIA Influence Subtype Selectivity between 32 and 34 Nicotinic Receptors via an Electrostatic Mechanism

Understanding the determinants of alpha-conotoxin (alpha-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a prerequisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide-rich peptides is difficult not only because of an absence of alpha-CTX/nAChR co-structures but also because it is challenging to predict how a mutation to an alpha-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the alpha-CTX LvIA that is 25-fold selective for the alpha 3 beta 2 nAChR over the related alpha 3 beta 4 nAChR subtype, which is a target for nicotine addiction. Using two-electrode voltage clamp electrophysiology, we identified LvIA[D11R] that is 2-fold selective for the alpha 3 beta 4 nAChR, reversing the subtype preference. This effect is specifically due to the change in charge and not shape of LvIA[D11R], as substitution of D11 with citrulline retains selectivity for the alpha 3 beta 2 nAChR. Furthermore, LvIA[D11K] shows a stronger reversal, with 4-fold selectivity for the alpha 3 beta 4 nAChR. Motivated by these findings, using site-directed mutagenesis, we found that beta 2[K79A] (I79 on beta 4), but not beta 2[K78A] (N78 on beta 4), largely restores the potency of basic mutants at position 11. Finally, to understand the structural basis of this effect, we used AlphaFold2 to generate models of LvIA in complex with both nAChR subtypes. Both models confirm the plausibility of an electrostatic mechanism to explain the data and also reproduce a broad range of potency and selectivity structure-activity relationships for LvIA mutants, as measured using free energy perturbation simulations. Our work highlights how electrostatic interactions can drive alpha-CTX selectivity and may serve as a strategy for optimizing the selectivity of LvIA and other alpha-CTXs.